1. Field of the Invention
The present invention relates to a process for the regeneration of sunflower plants from cell or tissue culture through embryogenesis and organogenesis. More specifically, cells or tissues of sunflower plants are cultured to produce calli. The calli can then be cultured for a prolonged period. The calli are then cultured on a preconditioning medium. The preconditioned calli are cultured to produce shoots which are cultured to produce roots whereby sunflower plants are produced. The present invention also relates to the sunflower plants and seeds which are produced by this method.
2. Description of the Prior Art
Several methods have been described in the prior art which result in the regeneration of sunflower. All of these methods have involved the use of organogenesis. However, these methods do not appear to be very efficient, and only result in the formation of a few regenerated plants. In organogenesis, plant parts are cultured on a first medium to induce callus formation. The callus can then be transferred to a second medium to induce shoot formation. The shoots are then transferred to a third medium to induce root formation, at which point the regenerated plantlets (plants) can be transferred to soil.
One example of inefficient organogenesis can be found in Sadu, Indian J. Exp. Biol., pages 110-111 (June 1974). Sadu reported the regeneration of sunflower by culturing stem pith tissue to form a callus which differentiated to form plantlets. The medium was the same throughout the regeneration scheme.
The medium used to form the callus and to differentiate into plants was a modified White's medium containing 1 ppm of the hormone indole acetic acid (IAA). The modified White's medium contained the following ingredients in one liter of medium:
______________________________________ Component Weight (mg) Component Weight (mg) ______________________________________ NH.sub.4 NO.sub.3 400 ZnSO.sub.4 2.7 KCl 65 thiamine 0.2 KNO.sub.3 80 nicotinic acid 0.5 KH.sub.2 PO.sub.4 12.5 pyridoxine 0.5 Ca(NO.sub.3).sub.2.H.sub.2 O 144 glycine 2.0 MgSO.sub.4.7H.sub.2 O 7.2 inositol 100 Na.sub.2 EDTA 25 sucrose 20000 H.sub.3 BO.sub.3 1.6 agar 6000 MnSO.sub.4.4H.sub.2 O 6.5 ______________________________________
It was found that 2,4-dichlorophenoxyacetic acid (2,4-D) promoted callus growth but did not support differentiation of the callus. The addition of 0.1 ppm kinetin resulted in callus growth but no differentiation.
Chandler and Jan describe the regneration of sunflower from immature embryos of interspecific crosses. The immature embryos are grown on growth medium as described by Chandler and Beard, below. After 2 to 3 weeks, the enlarged embryos are transferred to tubes containing MS medium (MS mineral salts, vitamins and sucrose) containing IAA and kinetin, generally used at 0.3 ppm and 1.0 ppm, respectively. The tubes are cultured at 28.degree. C. in the light to produce calli which then form shoots. Shoots are usually transferred to a MS medium containing no hormones but containing 0.2% activated charcoal to cause root formation. The plantlets are then transferred to soil.
Heaton discloses the regeneration of the domestic sunflower Helianthus annus L. from immature embryos. The immature embryos were plated on Gibco Murashige Shoot-Tip Rooting Medium. Callus formation was induced using 3% sucrose, 0.3 mg/l IAA and 1.0 mg/l kinetin. The embryos were kept in the dark for two weeks, then in the light for 12 hours per day. After five weeks, shoots formed on the callus. The shoots were transferred to rooting medium for plantlet production.
Binding et al, Z. Pflanazenphysiol. 101, 119 (1981) describe the regeneration of shoots of the sunflower Helianthus annus from protoplasts. Protoplasts were cultured in V-KM medium to form callus, and the calli were transferred to a low osmotic medium. The low osmotic medium comprises B5 medium with 15 .mu.M 6-benzylaminopurine (BA). Shoots formed on this medium.
Rogers et al, In Vitro 9, 463 (1974) describe the production of roots from a callus in Helianthus annus. This was performed by culturing plant tissue sequentially on three media. The first medium comprises MS basal medium with 1 mg/l .alpha.-naphthalene acetic acid (NAA), 1 mg/l kinetin and 5 mg/l 2,4-D. The second medium comprises MS basal medium with 0.5 mg/l kinetin and 2 mg/l IAA. The third medium comprises 0.05 mg./l kinetin and 0.1 mg/l IAA. No whole plants were produced by this method.
One additional method has been described to produce sunflower plants from an embryo. This method has found use in producing plants from embryos which do not develop in the original plant itself, either because of embryo abortion or seed dormancy resulting from the hybridization of non-compatible species. This method is embryo culture, and has been described by Chandler and Beard in The Sunflower, pages 45-47 (August/September 1980) and Crop Science 23, 1004 (1983). This process involves the rescue of the embryo prior to abortion followed by maturation of the embryo and germination to form the plant.
In this process, young embryos were isolated 3 to 7 days after pollination. These embryos were usually less than 0.1 mm in diameter. The embryos were plated on a solid, growth medium which contained B5 salts, vitamins, amino acids, the auxin .alpha.-naphthalene acetic acid (NAA) at a concentration of 0.05 mg/l, and 12% sucrose. It was found that if 9% sucrose was utilized instead of 12% and 1.0 mg/l indoleacetic acid (IAA) was used instead of NAA, then the young embryos has a tendency to grow as undifferentiated callus instead of embryos. In addition, very young embryos also germinated prematurely on this latter medium.
After 1 to 2 weeks, the enlarged (2-6 mm in diameter) embryos were transferred to a liquid medium for germination of the embryos to plants. During the enlargement period, some of the embryos began root formation and pigment synthesis. The embryos were matured to the cotyledon stage in order to obtain plant formation. The liquid, germination medium contained B5 salts and 1% sucrose. After the embryo generated roots and a shoot, it was transplanted to soil.
The present invention is the first instance of a general method for obtaining sunflower plants, i.e., regenerating sunflowers, through the use of embryogenesis and organogenesis. Sunflower plants and seeds are produced by this process. The sunflower plants resulting from this process may differ from the starting plant material as a result of somoclonal variation. The pathway is also useful in that it will enable the use of various selection processes to provide further variation. The plants which are produced can then be used in conventional breeding programs.